The present invention relates to analytical chemistry, and in particular relates to sample preparation for molecular analysis.
Depending upon needs, attributes, or other factors, much analytical chemistry can be divided (for purposes of discussion) into elemental analysis and molecular analysis. Elemental analysis is necessary and useful and incorporates tools that range from simple combustion and acid digestion to sophisticated instrument techniques such as (among many others) various forms of atomic spectroscopy and several different uses of x-rays.
Although 118 elements have been shown to exist, only 94 occur naturally on earth, and only 88 in greater than “extreme trace” quantities. Going further, 10 elements make up 99.8% of the earth's crust. Thus, the search and analysis for elements present in compositions is generally well defined.
In contrast, molecular analysis—the task of identifying one or more compounds in a sample—presents an enormously larger set of possibilities. The number of “naturally occurring” compounds (those produced by plants or animals) is immeasurably large, and the capabilities of modern organic and inorganic synthesis have generated—figuratively or literally—a similar number of synthetic compounds.
Of such immense numbers of compounds, the large majority (particularly of synthetics) are limited to laboratory use and academic interest. Nevertheless, many compounds remain for which identification or quantitative measurement or both are helpful or necessary. Even a small group of recognizable representative samples would include pesticides in food, other synthetic chemicals in food (antibiotics, hormones, steroids), synthetic compositions (benzene, toluene, refined hydrocarbons) in soil, and undesired compositions in everyday items (e.g., Bisphenol-A (“BPA”) in polycarbonate bottles and other plastic food packaging.
Identifying isolated molecular compounds is, using modern instrumentation, relatively straightforward. The typical tools include (but are not limited to) liquid or gas chromatography; visible, infrared, and ultraviolet spectroscopy; mass spectroscopy, and nuclear magnetic resonance (“NMR”). Once a molecular compound is identified, its concentration can often be determined based on known standards and calibration curves.
Because, however, these techniques require substantially pure isolated samples, some intermediate steps—generally referred to as “sample preparation”—must be carried out to isolate the compounds of interest (known or unknown) from the matrix (soil, plastic, food, etc.) in which they might be found and ready them for instrument analysis.
Based upon these and related factors, the market for molecular analysis is approximately 10 Limes that, of the market for elemental analysis.
In a general sense, extraction has been a main form of sample preparation; i.e., drawing one or more compounds of interest from a solid or a liquid (or a semi-solid) sample by mixing the sample with a solvent into which the desired compound(s) will move when given the opportunity.
For several generations (and continuing to date), sample preparation in the form of extraction has been carried out by the well-understood Soxhlet method which was invented in the 19th century Basically a single portion of solvent circulate repeatedly through a sample matrix until extraction is complete. To the extent the Soxhlet method has an advantage, it allows an extraction to continue on its own accord for as long as the boiling flask is heated and the condenser is cold.
In recent decades, advances in liquid chromatography have led to analogous uses of packed columns in a technique referred to as solid phase extraction (“SPE”). Originally, chromatography was used to separate fractions in mixed samples for analytical purposes, and indeed it still serves this purpose very well.
In SPE, the chromatography technique is modified to extract an analyte from a matrix. Nevertheless, SPE fundamentally remains a liquid chromatography technique in which molecules spread out (travel at different speeds) within a column based on their polarity, the particle size and polarity of the packed column (stationary phase), the polarity of the flowing liquid (mobile phase), the size (length and diameter) of the column and specific factors such as “hold-up volume.” “linear velocity,” and “flow rate.” See, e.g., Arsenault, J. C. 2012. Beginner's Guide to SPE. Milford Mass.: Waters Corporation. (Arsenault 2012).
Although SPE is useful, it has limiting characteristics, some of which include the following factors. First, a proper description of SPE is “liquid-solid phase extraction” because the sample matrix that holds the analyte is almost always a liquid.
Second, because SPE is essentially a liquid chromatography technique, it requires either column packing steps or a new column for each test, along with a potential pre-swelling step depending upon the material selected or required for the stationary phase. SPE typically requires different methods and manipulative steps for different analytes, and the packing must be very tight to allow proper flow and avoid channeling.
Third, SPE must match the mobile phase and the stationary phase (the sorbent in the packed column) to the expected characteristics of the analyte.
Fourth, a more deliberate (slower) flow through the packed column tends to produce better separation among the fractions. Thus, in a very real sense slower SPE is better than faster SPE.
As potential further disadvantages, in some pressurized methods, suitable extraction cells must be strong enough to withstand the vapor pressure of the vaporized solvent as well as that generated by any breakdown products from the sample. Depending upon the circumstances, such cells must be relatively thick which increases their heating and cooling times during an extraction cycle.
Some individual extraction cells are formed of several sub-items and can be difficult to assemble correctly, a critical step for safety purposes. In some cases the design and structure of instruments that use such cells are more complex, and thus generally more costly, and in some cases subject to more vigilant safety measures, which again increases cycle times and costs.
Additionally, when extraction cells are attached to single inlets and outlets, any kind of countercurrent flow such as viscous mixing or bubbling can become difficult or impossible.
Finally, if additional pressure (i.e., in addition to simple gravity flow) is required to move solvent through the SPE column, an external pump or vacuum pull must be applied, which in turn adds some lesser or greater amount of complexity to the system and technique.
More recently, a dispersive solid phase extraction (“dSPE”) method referred to as “QuEChERS” or “QuEChERS” (“quick-easy-cheap-effective-rugged-safe”) has become a standard for extraction preparation of molecular samples. Dispersive SPE addresses some of the disadvantages of SPE, but still requires an extraction step, the adjustment of pHI with an appropriate ionic salt, is labor-intensive (even if advantageous compared to other methods), and requires two separate centrifuge steps.
QuEChERS is in many ways less complex than Soxhlet extraction, but still requires a multi-step process. In the literature, this is sometimes called a “three step process” (e.g., Paragraph 0153 of U.S. Patent Application Publication No. 20160370357), but in reality QuEChERS requires at least the following: homogenization of the matrix that contains the analyte of interest; adding extraction solvent and loose sorbent particles; hand agitation; buffering; a second agitation step; a centrifuge separation step; decanting; dispersive solid phase extraction (“dSPE”) clean up; a second centrifuge separation step; and decanting the supernatant liquid following the centrifuge step.
In addition to the multi-step handling and transfer of the solvent, the sample, and the various mixtures, each of the centrifuge steps takes a recommended five minutes; so that the full QuEChERS sample preparation takes at least about 15-20 minutes. QuEChERS is also limited to room temperatures.
Accordingly, although the Soxhlet, SPE, and QuEChERS (dSPE) methods have their advantages, each remains relatively time-consuming. As a result, when multiple samples are required or desired to provide necessary or desired information, the time required to carry out any given extraction-based molecular preparation step reduces the number of samples that can be prepared in any given amount of time, thus reducing the amount of information available in any given time interval. To the extent that measurements, are helpful or necessary in a continuous process, this represents a longer gap between samples or before an anomalous or troublesome result can be identified.
In summary, among other disadvantages current sample preparation techniques are slow, require a large number of separate steps, use excess solvent, are difficult to automate, and operate under high liquid pressure.
Accordingly, a need continues to exist for efficient rapid extraction-based molecular preparation techniques.